This research proposal details our plan to establish the "proof of concept" that will support a subsequent R01 application to perfect and extend our methodology to expedite the functional and therapeutic analysis of hypothetical proteins identified by genomic sequencing. Accomplishing this challenging goal promises to provide an unprecedented wealth of information about cell biology, development, evolution and physiology that will have a significant impact on human health. The aim of this application is to further validate and increase the efficiency of our FAST-NMR (functional annotation screening technology by NMR) assay to assign a function to hypothetical proteins identified by the NIH-NIGMS Protein Structure Initiative (PSI). FAST-NMR uniquely combines structural biology, NMR ligand affinity screens and bioinformatics to discover biochemical functions or functional hypotheses of proteins of unknown function. The goal of the proposed research is to screen functionally annotated and hypothetical proteins with NMR structures determined by the Northeast Structural Genomics Consortium (NESG) to verify the accuracy of FAST-NMR functional assignments. Additionally, a high-performance affinity chromatography-mass spectrometry (HPAC-MS) component will be incorporated into the FAST-NMR screen to improve the efficiency and throughput of the assay. NESG proteins are associated with growth regulation and cancer, antibiotic resistance and other biomedically-important targets. Obtaining a functional assignment for these as of yet undiscovered proteins should provide key information for developing new therapies to treat various human diseases. The central hypothesis of our FAST-NMR assay is that proteins with similar function will have similar active-site structural characteristics, despite global differences in sequence and structure. A functional annotation is obtained by identifying similar active-site structure and sequence composition between proteins of known and unknown function. FAST-NMR is a further refinement of the generally accepted paradigm that global sequence or structure homology infers a similarity in function. Our proposed research is significant because the resultant FAST-NMR methodology will address an important need of PSI by providing an approach to annotate the expanding number of "orphaned" proteins deposited in the PDB whose function are currently unknown. The identification of novel therapeutic targets by screening NESG proteins in our FAST-NMR assay is invaluable for expediting the drug discovery process and improving human health. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to human health because accelerating the functional annotation of the vast number of novel proteins identified from genomic sequencing will lead to the discovery of new therapeutic targets and expedite the drug discovery process. The development of effective therapeutics is essential to addressing global public health problems associated with infectious disease, cancer and other illnesses.